Assessment of virus infection in cultured cells using metabolic monitoring

Singhvi, Rahul; Markusen, Julia F.; Ky, Bonnie; Horvath, Brian J.; Auniņš, John G.

doi:10.1007/bf00353926

Cited by 18 publications

(9 citation statements)

References 8 publications

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“…The culture is also characterized by a slight decrease of the ratio of lactate/glucose at 24 h post-infection. This is consistent with a previous report in which the increased lactate production of infected cells was used to assess the progression of virus infection in cultures (Singhvi et al, 1996).…”

Section: Cell Metabolism During Growth and Infection Of Perfusion Culsupporting

confidence: 64%

Insights into adenoviral vector production kinetics in acoustic filter‐based perfusion cultures

Henry

Dormond

Perrier

et al. 2004

Biotech & Bioengineering

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One of the major limitations in the production of adenoviral vectors is the reduction in cell-specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed-batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK-293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4-3 x 10(6) cell/mL, while maintaining a similar cell specific productivity (17,900 +/- 2400 VP/cell) to that of a batch infected at a low cell density (5 x 10(5) cell/mL). The highest virus concentrations (4.1 +/- 0.6 x 10(10) VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on-line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity.

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Section: Cell Metabolism During Growth and Infection Of Perfusion Culsupporting

confidence: 64%

Insights into adenoviral vector production kinetics in acoustic filter‐based perfusion cultures

Henry

Dormond

Perrier

et al. 2004

Biotech & Bioengineering

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“…Stimulation in glycolysis and increased respiration has been reported in adenovirus-infected cultures (Rozee et al, 1957;Williams et al, 1982). Infected cell metabolism was also studied in cultures infected with avian and porcine viruses, baculovirus, and varicella zoster virus (Kussow et al, 1995;Singhvi et al, 1996;Tom et al, 1995).…”

Section: Adenovirus-infected Perc6ா Cell Growth and Metabolismmentioning

confidence: 99%

Serum‐free suspension cultivation of PER.C6® cells and recombinant adenovirus production under different pH conditions

Xie

Pilbrough

Metallo

et al. 2002

Biotech & Bioengineering

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PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions. Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8. The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates. Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions. It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced. Adenovirus infection resulted in profound changes in cell growth and metabolism. Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection. Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH. The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study. Deviations from this optimum resulted in significant reductions of virus productivity. The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production.

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“…The prior studies either exploit features of viral infectious cycles, such as changes in cell metabolism and physical structure, or directly assay for the presence of virus or viral transgene. Several papers have reported monitoring changes in the respiration of infected cultures (6,(9)(10)(11) or changes in the rates of metabolite consumption (2,3,8,9). Two studies have looked at the green fluorescent protein as a marker transgene for studying infections (4,5), and one study has looked at the ABER capacitance probe as a tool for monitoring infections (7).…”

Section: Introductionmentioning

confidence: 98%

“…A few studies have concerned mammalian cell culture infections (2,6), but there seems to be only one study to date that has focused on adenovirus infections (4). The prior studies either exploit features of viral infectious cycles, such as changes in cell metabolism and physical structure, or directly assay for the presence of virus or viral transgene.…”

Section: Introductionmentioning

confidence: 98%

“…There have been a number of prior studies on monitoring of viral production processes (2)(3)(4)(5)(6)(7)(8)(9)(10)(11), most of which are concerned with the infection of Sf9 insect cell cultures. A few studies have concerned mammalian cell culture infections (2,6), but there seems to be only one study to date that has focused on adenovirus infections (4).…”

Section: Introductionmentioning

confidence: 99%

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Monitoring Adenovirus Infections with On-Line and Off-Line Methods

Monica¹,

Montgomery²,

Ayala³

et al. 2000

Biotechnol. Prog.

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Several known process monitoring methods were tested for their efficacy in the detection of adenovirus infections. The methods that we explored include several indirect indications of viral infections, including metabolic rate analysis, secondary gauges of respiration, cell size measurement, cell number and cell viability determination, and changes in capacitance. Direct indications of the adenovirus infection were also applied, including total viral particle and infectious particle measurements, as well as a flow cytometry method for detecting infected cells. All of the methods tested in the study provide some positive indication of an adenovirus infection. Many of the methods require repeated sampling, which may limit their utility in a manufacturing process. All of the indirect measures of viral infection may be limited by the fact that they do not uniquely identify an infection. The simplest monitoring methods appear to be detection of changes in respiration or the capacitance of the culture, both of which seem to provide a clear indication of an infection. Further work will be required to demonstrate that these indications are characteristic of only a successful and productive adenovirus infection.

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Assessment of virus infection in cultured cells using metabolic monitoring

Cited by 18 publications

References 8 publications

Insights into adenoviral vector production kinetics in acoustic filter‐based perfusion cultures

Insights into adenoviral vector production kinetics in acoustic filter‐based perfusion cultures

Serum‐free suspension cultivation of PER.C6® cells and recombinant adenovirus production under different pH conditions

Monitoring Adenovirus Infections with On-Line and Off-Line Methods

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